1. Field of the Invention
The present invention relates to proteins and nucleic acids related to salt tolerance in plants.
2. Description of the Background
Soil salinity is a major abiotic stress for plant agriculture. Sodium ions in saline soils are toxic to plants due to its adverse effects on K+ nutrition, cytosolic enzyme activities, photosynthesis and metabolism (1, 2). Three mechanisms function cooperatively to prevent the accumulation of Na+ in the cytoplasm, i.e. restriction of Na+ influx, active Na+ efflux and compartmentation of Na+ in the vacuole (1). The wheat high-affinity K+ transporter HKT1 functions as a Na+xe2x80x94K+ cotransporter, which confers low-affinity Na+ uptake at toxic Na+ concentrations (3). Thus HKT1 could represent one of the Na+ uptake pathways in plant roots. The low-affinity cation transporter LCT1 from wheat may also mediate Na+ influx into plant cells (4). In addition, patch clamp studies have shown that non-selective cation channels play important roles in mediating Na+ entry into plants (5). The Arabidopsis thaliana AtNHX1 gene encodes a tonoplast Na+/H+ antiporter and functions in compartmentalizing Na+ into the vacuole (6). Over-expression of AtNHXl enhances the salt tolerance of Arabidopsis plants (7).
No Na+ efflux transporter has been cloned from plants. Plants do not appear to have a Na+-ATPase at the plasma membrane (1). It is expected that proton motive force created by H+-ATPases would drive Na+ efflux from plant cells through plasma membrane Na+/H+ antiporters (8). Fungal cells contain both Na+-ATPases and Na+/H+ antiporters at the plasma membrane. In the yeast Saccharomyces cerevisiae, plasma membrane Na+xe2x80x2-ATPases play a predominant role in Na+ efflux and salt tolerance (9). In contrast, Na+/H+ antiporters are more important for Na+ efflux and salt tolerance in the fungus Schizosaccharomyces pombe (10).
Recently, several Arabidopsis sos (for salt overly sensitive) mutants defective in salt tolerance were characterized (11,12,13). The sos mutants are specifically hypersensitive to high external Na+ or Li+ and also unable to grow under very low external K+ concentrations (13). Allelic tests indicated that the sos mutants define three SOS loci, i.e., SOS1, SOS2 and SOS3 (13). The SOS3 gene encodes an EF-hand type calcium-binding protein with similarities to animal neuronal calcium sensors and the yeast calcineurin B subunit (14). In yeast, calcineurin plays a central role in the regulation of Na+ and K+ transport. Mutations in calcineurin B lead to increased sensitivity of yeast cells to growth inhibition by Na+ and Li+ stresses (15). The SOS2 gene was recently cloned and shown to encode a serine/threonine type protein kinase (16). Interestingly, SOS2 physically interacts with and is activated by SOS3 (17). Therefore, SOS2 and SOS3 define a novel regulatory pathway for Na+ and K+ homeostasis and salt tolerance in plants. The SOS3/SOS2 pathway has been predicted to control the expression and/or activity of ion transporters (17). However, the identities of the transporters regulated by this pathway are not known.
Among the three SOS loci, SOS1 plays the greatest role in plant salt tolerance. Compared to sos2 and sos3 mutant plants, sos1 mutant plants are even more sensitive to Na+ and Li+ stresses (13). Double mutant analysis indicated that SOS1 functions in the same pathway as SOS2 and SOS3 (12, 13). Thus, SOS1 may be a target for regulation by the SOS3/SOS2 pathway.
Accordingly, there remains a need in the art to isolate the SOS1 gene and the protein encoded thereby.
Furthermore, because of limited water supplies and the widespread use of irrigation, the soils of many cultivated areas have become increasingly salinized. In particular, modern agricultural practices such as irrigation impart increasing salt concentrations when the available irrigation water evaporates and leaves previously dissolved salts behind. As a result, the development of salt tolerant cultivars of agronomically important crops has become important in many parts of the world. For example, in salty soil found in areas such as Southern California, Arizona, New Mexico and Texas.
Dissolved salts in the soil increase the osmotic pressure of the solution in the soil and tend to decrease the rate at which water from the soil will enter the roots. If the solution in the soil becomes too saturated with dissolved salts, the water may actually be withdrawn from the plant roots. Thus the plants slowly starve though the supply of water and dissolved nutrients may be more than ample. Also, elements such as sodium are known to be toxic to plants when they are taken up by the plants.
Salt tolerant plants can facilitate use of marginal areas for crop production, or allow a wider range of sources of irrigation water. Traditional plant breeding methods have, thus far, not yielded substantial improvements in salt tolerance and growth of crop plants. In addition, such methods require long term selection and testing before new cultivars can be identified.
Accordingly, there is a need to increase salt tolerance in plants, particularly those plants which are advantageously useful as agricultural crops.
The present invention is based, in part, on the isolation of the SOS1 locus through positional cloning. It is predicted to encode a transmembrane protein with similarities to plasma membrane Na+/H+ antiporters from bacteria and fungi. The results of the present invention suggest that a plasma membrane-type Na+/H+ antiporter is essential for plant salt tolerance. The steady state level of SOS1 transcript is up-regulated by NaCl stress. The sos2 mutation abolishes SOS1 up-regulation in the shoot. In the sos3 mutant, no SOS1 up-regulation is found in the shoot or root. Therefore, SOS1 gene expression under NaCl stress is controlled by the SOS3/SOS2 regulatory pathway.
Accordingly, the present invention provides an isolated polynucleotide which encodes a protein comprising the amino acid sequence of SEQ ID NO:2.
In a preferred embodiment the polypeptide has Na+/H+ transporter activity.
In another preferred embodiment the polynucleotide comprises SEQ ID NO: 1, polynucleotides which are complimentary to SEQ ID NO:1, polynucleotides which are at least 70%, 80% and 90% identical to SEQ ID NO:1; or those sequence which hybridize under stringent conditions to SEQ ID NO:1, the stringent conditions comprise washing in 5xc3x97SSC at a temperature from 50 to 68xc2x0 C.
In another preferred embodiment the polynucleotides of the present invention are in a vector and/or a host cell. Preferably, the polynucleotides are in a plant cell or transgenic plant. Preferably, the plant is Arabidopsis thaliania or selected from the group consisting of wheat, corn, peanut cotton, oat, and soybean plant. In a preferred embodiment, the polynucleotides are operably linked to a promoter, preferably an inducible promoter.
In another preferred embodiment the present invention provides, a process for screening for polynucleotides which encode a protein having Na+/K+transporter activity comprising hybridizing the polynucleotide of the invention to the polynucleotide to be screened; expressing the polynucleotide to produce a protein; and detecting the presence or absence of Na+/K+transporter activity in said protein.
In another preferred embodiment, the present invention provides a method for detecting a nucleic acid with at least 70% homology to nucleotide SEQ ID NO:1, sequences which are complimentary to SEQ ID NO:1 and/or which encode a protein having the amino acid sequence in SEQ ID NO:2 comprising contacting a nucleic acid sample with a probe or primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of claim 1, or at least 15 consecutive nucleotides of the complement thereof.
In another preferred embodiment, the present invention provides a method for producing a nucleic acid with at least 70% homology to the polynucleotides of the present invention comprising contacting a nucleic acid sample with a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of claim 3, or at least 15 consecutive nucleotides of the complement thereof.
In another preferred embodiment, the present invention provides a method for making SOS2 protein, comprising culturing the host cell carrying the polynucleotides of the invention for a time and under conditions suitable for expression of SOS2, and collecting the SOS2 protein.
In another preferred embodiment, the present invention provides a method of making a transgenic plant comprising introducing the polynucleotides of the invention into the plant.
In another preferred embodiment, the present invention provides method of increasing the salt tolerance of a plant in need thereof, comprising introducing the polynucleotides of the invention into said plant.
In another preferred embodiment, the present invention provides an isolated polypeptide comprising the amino acid sequence in SEQ ID NO: 2 or those proteins that are at least 70%, preferably 80%, preferably 90% and preferably 95% identity to SEQ ID NO:2. Preferably, the polypeptides have Na+/K+transporter activity.